The overall objective for Project 1 is to enhance the immunogenicity and efficacy of rAAV/HIV vaccines. We have shown that a single dose of a first generation SlV vaccine based on rAAV serotype 2 (rAAV2) vectors is highly immunogenic in rhesus macaques. These same vaccines significantly reduced viral burden in macaques after challenge with pathogenic SIV. More recently, we have shown that another serotype (rAAV1) is 1- 2 orders of magnitude better at gene delivery to muscle than rAAV2. These observations suggest that: (i) the current first generation vaccine is probably sub-optimal; (ii) alternate serotype vectors might allow for dramatic increases in transduction (and immunogenicity); and, (iii) alternate serotype vectors offer the possibility of"prime-boost" regimens where the induction of vector cross-neutralizing antibodies is avoided. In Specific Aim 1, we will extend these observations by testing prime-boost regimens using alternate serotypes (rAAV1 and rAAV5) that are more efficient than rAAV2 at gene transfer and are genetically distinct in the capsid gene (no cross-reacting antibodies). We will also examine the effects of pre-existing immunity to AAV on the immunogenicity of rAAV/SIV (and HIV) vaccines in macaques. In addition to vector enhancements, we have tested "non-traditional" molecular adjuvants to augment immune responses engendered by rAAV vaccines and have identified promising candidates. In Specific Aim 2, we will advance these candidates into immunogenicity trials in macaques. Building on the data from this Project and Projects 2 and 3, we will design and test a second generation vaccine in immunogenicity and challenge experiments in macaques. A final challenge trial will be combined with a trial proposed in Project 4 wherein the second generation rAAV vaccine will be combined with an rAAV vector that directs the expression of antibody genes that neutralize primary isolates of HIV- 1.